Dataset Information

Two novel, putatively cell wall-associated and glycosylphosphatidylinositol-anchored alpha-glucanotransferase enzymes of Aspergillus niger.

ABSTRACT: In the genome sequence of Aspergillus niger CBS 513.88, three genes were identified with high similarity to fungal alpha-amylases. The protein sequences derived from these genes were different in two ways from all described fungal alpha-amylases: they were predicted to be glycosylphosphatidylinositol anchored, and some highly conserved amino acids of enzymes in the alpha-amylase family were absent. We expressed two of these enzymes in a suitable A. niger strain and characterized the purified proteins. Both enzymes showed transglycosylation activity on donor substrates with alpha-(1,4)-glycosidic bonds and at least five anhydroglucose units. The enzymes, designated AgtA and AgtB, produced new alpha-(1,4)-glycosidic bonds and therefore belong to the group of the 4-alpha-glucanotransferases (EC 2.4.1.25). Their reaction products reached a degree of polymerization of at least 30. Maltose and larger maltooligosaccharides were the most efficient acceptor substrates, although AgtA also used small nigerooligosaccharides containing alpha-(1,3)-glycosidic bonds as acceptor substrate. An agtA knockout of A. niger showed an increased susceptibility towards the cell wall-disrupting compound calcofluor white, indicating a cell wall integrity defect in this strain. Homologues of AgtA and AgtB are present in other fungal species with alpha-glucans in their cell walls, but not in yeast species lacking cell wall alpha-glucan. Possible roles for these enzymes in the synthesis and/or maintenance of the fungal cell wall are discussed.

SUBMITTER: van der Kaaij RM

PROVIDER: S-EPMC1951109 | biostudies-literature | 2007 Jul

REPOSITORIES: biostudies-literature

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Two novel, putatively cell wall-associated and glycosylphosphatidylinositol-anchored alpha-glucanotransferase enzymes of Aspergillus niger.

van der Kaaij R M RM Yuan X-L XL Franken A A Ram A F J AF Punt P J PJ van der Maarel M J E C MJ Dijkhuizen L L

Eukaryotic cell 20070511 7

In the genome sequence of Aspergillus niger CBS 513.88, three genes were identified with high similarity to fungal alpha-amylases. The protein sequences derived from these genes were different in two ways from all described fungal alpha-amylases: they were predicted to be glycosylphosphatidylinositol anchored, and some highly conserved amino acids of enzymes in the alpha-amylase family were absent. We expressed two of these enzymes in a suitable A. niger strain and characterized the purified pro ...[more]

PMID: 17496125

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Project description:In order to produce therapeutic glycoproteins modified with human-like N-glycan structures, much progress has been achieved toward the humanization of N-glycosylation pathways in yeasts. In this study, a Pichia pastoris mutant Glyco4, which has a humanized N-glycosylation pathway and could successfully generate the human-like N-glycans, was carefully characterized. Glyco4 displays a significant growth delay and cell wall defects. Comparative transcriptomics reveals that manipulating the N-glycosylation pathway could notably affect the expression pattern of numerous biological pathways in Glyco4. Among the differentially expressed genes, the down-regulation of PpSPI1 was proven to be the main cause of the cell wall defects in Glyco4. Deletion of PpSPI1 in P. pastoris GS115 strain presented growth delay and weaker resistance to cell wall/membrane perturbing agents. PpSpi1 was shown to be a glycosylphosphatidylinositol-anchored cell wall glycoprotein and involved in the formation of the mannoprotein layer on the outer surface of cell wall. Overexpressing PpSPI1 in Glyco4 could partially recover the cell wall defects and also improve its resistance to cell wall perturbing agents and osmotic stress. Thus, overexpression of PpSpi1 is a useful strategy to facilitate P. pastoris industrial applications in the manufacture of human glycoproteins. IMPORTANCE Engineering of biological pathways in various microorganisms is a promising direction for biotechnology. Since the existing microbial cells have evolved over a long period of time, any artificial engineering may cause some unexpected and harmful effects on them. Systematically studying and evaluating these engineered strains are very important and necessary. In order to produce therapeutic proteins with human-like N-glycan structures, much progress has been achieved toward the humanization of N-glycosylation pathways in yeasts. The properties of a P. pastoris strain with humanized N-glycosylation machinery were carefully evaluated in this study. Our work has identified a key glycoprotein (PpSpi1) responsible for the poor growth and morphological defects of this glycoengineered strain. Overexpression of PpSpi1 could significantly rescue the growth defect of the glycoengineered P. pastoris and facilitate its future industrial applications.

Dataset Information

Two novel, putatively cell wall-associated and glycosylphosphatidylinositol-anchored alpha-glucanotransferase enzymes of Aspergillus niger.

Publications

Two novel, putatively cell wall-associated and glycosylphosphatidylinositol-anchored alpha-glucanotransferase enzymes of Aspergillus niger.

Similar Datasets

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